The invention relates to a method and a device for feeding a liquid sample into the capillary of a capillary electrophoresis apparatus.
Electrophoresis is an electrochemical method by which it is possible to separate electrically charged particles, and by certain special methods also uncharged particles, in an electrolyte solution, the size of the particles varying from the smallest ions and molecules to colloidal particles. The particles migrate in an electric field at different speeds depending on their electrical charge and other properties. In zone electrophoresis, the sample to be separated is placed as a narrow zone in the electrolyte solution, i.e. background solution. In the electric field the various components of the sample become separated as separate zones. In order for the zones not to spread excessively, convection of the background solution must be inhibited. In zone electrophoresis this is done, for example, by stabilizing the solution by paper, gel or some other supporting medium.
The latest stage of electrophoresis development is capillary electrophoresis, which is one of the most rapidly progressing areas of application in analytical chemistry. In this method the background solution is in a narrow or tube, capillary, which is so narrow that the viscous forces of the liquid will inhibit convection. The inner diameter of the capillary usually ranges from 20 to 100 .mu.m. Electrophoresis is thus performed in a free solution; thereby disturbances caused by a supporting medium are eliminated. It is also easy to eliminate from the capillary the thermal energy generated by the electric current, and thus a high electric field can be applied, thus speeding up separation. Furthermore, automation of capillary electrophoresis is easy.
In capillary zone electrophoresis, two reservoirs containing the background electrolyte solution are interconnected by a capillary tube which contains the same solution. Each reservoir is equipped with an electrode. The sample to be analyzed is introduced as a short zone into one end of the capillary. For the introduction of a sample the end of the capillary is usually transferred into one reservoir, and the desired amount of the sample solution is injected into the capillary, where-after the capillary end is transferred back into the background solution. By means of electrodes in the reservoirs, an electric field is applied on the capillary, usually ranging from 200 to 1000 V/cm, under the effect of which the electrically charged particles will begin to move in the capillary. The different particles will separate from each other if they have different speeds in the electric field. The particle zones will pass a detector at the other end of the capillary at different times, and their signals are measured.
Capillary electrophoresis has many advantages, such as high separation speed, high resolution, and small required sample size. In order to achieve high resolution it is important that the sample can be introduced as a zone as short as possible into the capillary. The capillary volume in itself is very small, and the sample volume must be only a small proportion of the capillary volume. The problem of sample injection in capillary electrophoresis has not been solved satisfactorily. At present there are two methods used for sample introduction, the hydrodynamic method and the electrokinetic method. In both methods the capillary is first filled with a background solution, and for the time of sample injection the samplereceiving end of the capillary is transferred into the sample solution, the amount of which must be sufficient for the capillary end to be immersed in it.
In the hydrodynamic method, the sample is injected into the capillary by a pressure difference. The pressure difference is produced either by placing the capillary ends at different levels, whereby a hydrostatic pressure difference is produced, or in a sealable sample reservoir overpressure is generated by means of gas, the overpressure injecting the sample solution into the capillary. The amount of sample passing into the capillary is controlled by the selection of the pressure difference and its effective time.
In electrokinetic introduction of a sample, a capillary end is placed in the sample reservoir and the electrophoresis current is applied between the electrodes, whereupon the charged particles in the sample will begin to travel into the capillary at speeds depending on their specific mobilities. Furthermore, the entire sample solution will begin to flow into the capillary under the effect of electro-osmosis. The sample amount transferred is a function of the electric field and the time, and it is different for substances with different electric mobilities.
These known sample-introduction methods presuppose that the sample-receiving end of the capillary is transferred from the background solution into the sample solution, and after sampling back into the background solution, and that there is a sufficient amount of the sample. The transfer steps are inconvenient and affect the injected sample amount, and additionally they are to some extent uncontrollable. The mere dipping of the capillary in the sample solution will cause a small amount of sample to be injected. In order for the introduction of the sample to be precisely controllable, it must be performed without moving the capillary out of the background solution.
U.S. Pat. No. 5,141,621 discloses a capillary electrophoresis system wherein the sample-injection capillary and the separation capillary of the electrophoresis apparatus are fixedly linked to an interface chamber. The construction of the interface is difficult and the fixed interface complicates, for example, the replacement of the injection capillary and/or the separation capillary. Furthermore, in the system of the U.S. patent, it is necessary to use pumping for pumping the sample solution through the injection capillary into the interface chamber or into the separation capillary. In this case a much larger amount of the sample is needed than what is introduced into the separation capillary.